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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2006 October 25 - 2007 January 30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to EC and OECD guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
See below.
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
See below.
Principles of method if other than guideline:
Deviations:
1. One adrenal from animal no. 67 and from animal no. 4 was not available for histopathology. Reasons for this included that these tissues were not discernable at necropsy or trimming, or were erroneously not collected at necropsy. Tissues are listed in raw data and pathology report. Evaluation: Sufficient tissues were available for evaluation.
2. No terminal body weight was determined from animal no. 55, and no adrenal weight was recorded for animal no. 4. Evaluation: Sufficient data were available for evaluation.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Toluene-4-sulphonamide
EC Number:
200-741-1
EC Name:
Toluene-4-sulphonamide
Cas Number:
70-55-3
Molecular formula:
C7H9NO2S
IUPAC Name:
4-methylbenzenesulfonamide
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): p-TSA
- Substance type: White crystalline powder
- Physical state: Solid
- Analytical purity: 99.9%
- Impurities (identity and concentrations): 0.1% water
- Purity test date: 2006 July 28
- Lot/batch No.: 0603514009
- Expiration date of the lot/batch: 2008 March 31
- Stability under test conditions: Stable in diet for at least 36 days at room temperature
- Storage condition of test material: At room temperature in the dark in tightly closed containers. Keep dry.
- Other: Hygroscopic. Reacts with acids with formation of heat.

Test animals

Species:
rat
Strain:
other: Wistar Han, Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Housing: 5 animals/sex in Macrolon cages with sterilised saw dust as bedding material and paper as cage-enrichment.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0-23.2
- Humidity (%): 35-87
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): diets were prepared for weeks 1-4, 5-8 and 9-13.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analysed in the diets were between 83 and 113% of target. In the control diet, no test substance was detected.
Duration of treatment / exposure:
At least 90 days.
Frequency of treatment:
Ad libitum
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
3000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on results of a 28-day range finding study

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, mortality
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-test and week 13
- Dose groups that were examined: all animals, animals from groups 1 and 4, respectively

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: Erythrocytes count, haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelet count, Red cell distribution width, Total leucocytes count, Differential leucocyte count, Prothrombin time, Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: Alanine aminotransferase, Alkaline phosphatase, Aspartate aminotransferase, Bilirubin, total, Chloride, Cholesterol, total, Creatinine, Glucose, Phosphorus, Protein, total, Protein, albumin, Urea, Calcium, Potassium, Sodium.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 12-13
- Dose groups that were examined: all
- Battery of functions tested: grip strength / motor activity / other: hearing ability, pupillary reflex, static righting reflex.
Sacrifice and pathology:
Organ weights: Adrenal glands, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus, Liver.
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Other examinations:
None.
Statistics:
None.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain were reduced in males and females at 10.000 ppm, achieving a level of statistical significance on most occasions. Total weight gain deficit over the treatment period compared to controls was 21% and 11% for males and females, respectively.
Body weights and body weight gain of animals at 1000 or 3000 ppm remained in the same range as controls over the study period.

HISTOPATHOLOGY: NON-NEOPLASTIC
In 2/10 males at 10.000 ppm, a minimal degree of hyperplasia of the urothelium of the urinary bladder was recorded.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Basis for effect level:
other: Reduced body weight gain, histopathology (minimal urethelial hyperplasia in two animals)
Dose descriptor:
NOAEL
Effect level:
214 mg/kg bw/day (actual dose received)
Sex:
male
Dose descriptor:
NOAEL
Effect level:
248 mg/kg bw/day (actual dose received)
Sex:
female
Dose descriptor:
LOAEL
Effect level:
10 000 ppm
Dose descriptor:
LOAEL
Effect level:
738 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Reduced body weight gain, histopathology (minimal urethelial hyperplasia in two animals)
Dose descriptor:
LOAEL
Effect level:
795 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Reduced body weight gain.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
LOAEL: 10.000 ppm (738 mg/kg/day for males, or 795 mg/kg/day for females).
NOAEL: 3000 ppm (214 mg/kg/day for males, or 248 mg/kg/day for females).
Executive summary:

The study was based on the following guidelines.

- EC Directive 67/548/EEC, B Repeated Dose (90 days) Toxicity (oral), 2001.

- OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, 1998.

- EPA 712-C-98-199, 90-Day Oral Toxicity, 1998.

Based on a 28-day dietary range finding study (NOTOX Project 474053) and in consultation with the sponsor, the dose levels for this 90-day dietary study were selected to be 0, 1000, 3000 and 10.000 ppm. SPF-bred Wistar Han rats received the test substance by dietary intake for at least 90 days. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Chemical analyses of diet preparations were conducted to assess accuracy and homogeneity of diet preparations.

The following parameters were evaluated: Clinical signs (daily), functional observations (week 13), body weight and food consumption (weekly), and ophthalmoscopy (pretest and week 13). At termination: clinical pathology, macroscopy, organ weights and histopathology on a selection of tissues.

RESULTS

Homogeneity and accuracy of diet preparations were demonstrated by analyses.

Dietary inclusion levels of 0, 1000, 3000 and 10.000 ppm were equivalent to an average test article intake of 0, 70, 214 and 738 mg/kg body weight/day for males, and 0, 80, 248 and 795 mg/kg body weight/day for females.

At 10.000 ppm, a significant and consistent reduction of body weight gain was recorded (up to 21% compared to control animals).

Histopathology revealed a minimal degree of hyperplasia of the urothelium of the urinary bladder in two males at 10.000 ppm.

No other treatment-related toxicologically significant changes were noted in any of the remaining parameters examined/determined in this study (i.e. clinical appearance, functional observations, food consumption, clinical laboratory investigations, macroscopic examination and organ weights).

The observed minimal urethelial hyperplasia in two animals seems to be caused by local irritation by the chronic availability of p-TSA metabolite (p-sulphamonylbenzoic acid) in the urine, following the rapid and complete excretion of p-TSA. Such effect is local and fully reversible when exposure ends. As the hyperplasia is observed in only two animals, is minimal and likely a fully reversible temporal effect upon exposure, is not an adverse effect persé, the reduction of the body weight gain of 21% in males is considered serious enough to assign the 10000 ppm a LOAEL. Especially as a relation to palatability is not completely clear.

LOAEL: 10.000 ppm (738 mg/kg/day for males, or 795 mg/kg/day for females).

NOAEL: 3000 ppm (214 mg/kg/day for males, or 248 mg/kg/day for females).